Anti-obesity agents

ABSTRACT

Disclosed are anti-obesity agents comprising 3-ketosteroidal compounds as defined in the specification. The present invention also discloses pharmaceutical compositions and use of these compounds in the prevention and treatment of obesity.

This application is a 371 of PCT/JP94/01631 filed Sep. 30, 1994.

TECHNICAL FIELD

The present invention relates to anti-obesity agents, more specificallyto anti-obesity agents which comprise as an active ingredient3-ketosteroid compounds having ketone groups at the C₃ position of thecholestane skeletons.

BACKGROUND OF THE INVENTION

Obesity is a condition under which the proliferation of tissues ofbody-composing lipids is increased abnormally. This abnormal conditionis produced when energy intake is continuously greater than energyconsumption, with the resulting excess energy being converted intoneutral lipid which is accumulated in lipid tissues.

Obesity is important as a risk factor of the onset of diseasesrepresented by geriatric diseases from hygienic and cosmetic viewpoints.Harmful influences of obesity have been recognized for a long time inadvanced nations. Agents for preventing and/or treating obesity whichhave been developed until now have side effects or produceunsatisfactory effects.

JP A 5-170651 discloses that anti-obesity agents which comprise4-cholestene-3-one as an active ingredient have a body lipid-inhibitingeffect and present little or no toxicity. However, there still exists aneed for more effective anti-obesity agents with less or no side effectsand lower toxicity.

Therefore, an object of the present invention is to provide moreeffective anti-obesity agents with less or no side effects and lowertoxicity. Another object of the present invention is to provide foodproducts which are useful for preventing and/or treating obesity.

DISCLOSURE OF THE INVENTION

The inventors found that 3-ketosteroid compounds having ketone groups atthe C₃ position of the cholestane skeletons produce a high anti-obesityeffect and have less or no side effects and lower toxicity. The presentinvention was accomplished on the basis of these findings. The presentinvention provides anti-obesity agents which comprise 3-ketosteroidcompounds as an active ingredient. The present invention also providesfood products which contain anti-obesity agents comprising 3-ketosteroidcompounds as an active ingredient.

The present invention further provides pharmaceutical compositions whichcomprise 3-ketosteroid compounds as an active ingredient andpharmaceutically acceptable carriers.

The present invention also provides a method for preventing and/ortreating obesity which comprises administering an effective amount of a3-ketosteroid compound to a human.

The anti-obesity agents of the present invention show substantially notoxicity and hence are useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course of the average body weights of CDF1 mice invarious experimental groups.

FIG. 2 shows the average intraperitoneal lipids in CDF1 mice in variousexperimental groups at the end of feeding.

FIG. 3 shows the time course of the average body weight of CDF1 micetreated with 6-hydroxy-4-cholestene-3-one.

FIG. 4 shows the average intraperitoneal lipid in6-hydroxy-4-cholestene-3-one-treated CDF1 mice at the end of feeding.

FIG. 5 shows the time course of the average body weight of CDF1 micetreated with 4-cholestene-3,6-dione.

FIG. 6 shows the average intraperitoneal lipid in CDF1 mice treated with4-cholestene-3,6-dione.

FIG. 7 shows the average intraperitoneal lipid in SD rats treated with5-cholestene-3-one.

BEST MODE FOR CARRYING OUT THE INVENTION

The anti-obesity agents of the present invention comprise 3-ketosteroidcompounds as an active ingredient. Any 3-ketosteroid compounds can beused, provided that they have an anti-obesity effect. Preferred is atleast one compound selected from the group consisting of5-cholesten-3-one, 5 β-cholestan-3-one, cholesta-4,6-dien-3-one, 6β-bromo-4-cholesten-3-one, 6-hydroxy-4-cholesten-3-one and4-cholestene-3,6-dione. 5-Cholesten-3-one, 6-hydroxy-4-cholesten-3-oneand 4-cholestene-3,6-dione are more preferred as having a higheranti-obesity effect.

5-Cholesten-3-one, 5 β-cholestan-3-one, cholesta-4,6-dien-3-one, 6β-bromo-4-cholesten-3-one, 6-hydroxy-4-cholesten-3-one and4-cholestene-3,6-dione, which can be used in the present invention, arerepresented by the following formulae. ##STR1##

5-Cholesten-3-one, 5 β-cholestan-3-one, cholesta-4,6-dien-3-one, 6β-bromo-4-cholesten-3-one, 6-hydroxy-4-cholesten-3-one and4-cholestene-3,6-dione may be synthesized chemically, producedbiologically by culturing a microorganism capable of producing a desiredcompound, or synthesized with an enzyme derived from a microorganism.Alternatively, commercially available ones produced by any one of thesemethods may be used. For example, 5-cholesten-3-one can be producedusing cholesterol as a starting material by the method of Cheng et al.(Y.-s. Cheng, W. L. Liu and S.-h. Chen, Synthesis; 1980, 223) or using4-cholesten-3-one as a starting material by the method of Ringold et al.(H. J. Ringold and S. K. Malhotra, Tetrahedron Lett., 1962, 669). 5β-Cholestan-3-one can be produced using 4-cholesten-3-one as a startingmaterial by the method of Tsuji et al. (N. Tsuji, J. Suzaki, and M.Shiota, J. Org. Chem., 45: 2729, 1980). Cholesta-4,6-dien-3-one can beproduced using 4-cholesten-3-one as a starting material by a combinationof the method of Chowdhury et al. (P. K. Chowdhury, R. P. Sharma, and J.N. Barua, Tetrahedron Lett., 24: 3383, 1983) and that of Minami et al.(I. Minami, K. Takahashi, I. Shimizu, and J. Tsuji, Tetrahedron, 42:2971, 1986). 6 β-Bromo-4-cholesten-3-one can be produced by reacting thedienol acetate prepared by the method of Chowdhury et al. (ibid.) withbromine in a dioxane-phosphate buffer (pH 7.0).6-Hydroxy-4-cholesten-3-one can be produced by reacting pyridiniumchlorochromate with 5,6-epoxycholesterol. 4-Cholestene-3,6-dione can beproduced using 4-cholesten-3-one as a starting material by a combinationof the method of Hevl and Herr (F. W. Hevl and M. E. Herr, J. Am. Chem.Soc., 75: 1918, 1953) and that of Malhotra et al. (S. K. Malhotra, J. J.Hostynek and A. F. Lundin, J. Am. Chem. Soc., 90: 6565, 1968).

While not being limited to a specific theory, it is assumed that the3-ketosteroid compounds produce the anti-obesity effect by any one ormore of the following mechanisms.

(1) Inhibition of the formation of bile acid micelles in the intestinesand competition against the dissolving of lipids in bile acid micelles.

(2) Inhibition of the formation of lipoprotein membranes in intestinaltissues and competition against the incorporation of lipids intolipoproteins.

(3) Inhibition of the synthesis of cholesterols in the livers andcompetition against the effects of cholesterols.

The anti-obesity agents of the present invention can be added to foodproducts. In this case, the 3-ketosteroid compounds may be addeddirectly to food products. Alternatively, the 3-ketosteroid compoundsmay be added to fats and/or oils and the resulting mixture may be usedto produce food products. Examples of the food products include anyfoods and drinks including natural and processed products, feeds fordomestic animals and cultured fish and the like. The 3-ketosteroidcompounds can be added in amounts of 1-5,000 mg to 100 g of a foodproduct or fat and/or oil. The anti-obesity agents of the presentinvention may be formulated in the form of solutions, suspensions,powders, granules, capsules or the like and the formulated preparationsmay be added to food products or fats and/or oils.

The anti-obesity agents of the present invention can be formulated inpharmaceutical compositions for use. In this case, the route ofadministration of the pharmaceutical compositions includes but is notlimited to oral, intravenous, intraperitoneal, subcutaneous andintramuscular administrations. Oral administration is preferred. In thecase of oral administration, the 3-ketosteroid compounds may beadministered with or without pharmaceutically acceptable carriers in theform of solutions, suspensions, powders, granules, tablets, capsules orthe like. Examples of the carriers include any conventionally used ones,for example, sugars such as lactose, sucrose and glucose, starch,inorganic compounds such as calcium carbonate and calcium sulfate,crystalline cellulose, distilled water, purified water, sesame oil,soybean oil, corn oil, olive oil, cotton seed oil and the like. Informulating the pharmaceutical compositions, additives such as binders,lubricants, dispersers, suspending agents, emulsifying agents, diluents,buffering agents, antioxidants, bacteriostats and the like may be used.In the case where the pharmaceutical compositions are used asinjections, an appropriate buffering agent, tonicity agent and the likemay be added and the resulting mixtures may be dissolved in oils such asvegetable oils. The pharmaceutical compositions may be used in admixtureor combination with other medicaments. The pharmaceutical compositionsmay be sterilized.

The dose of the anti-obesity agents of the present invention can bevaried depending on the age and sex of the patient, severity of thedisease to be treated, the route of administration, the number ofadministrations, the dosage form and the like. In the case of oraladministration, the dose of the 3-ketosteroid compounds is appropriatelyin the range from 1 to 1,000 mg/kg body weight/day for adult patients.

The present invention will be explained in greater detail with referenceto the following examples, which are intended to be illustrative of butnot limiting the scope of the present invention.

PREPARATION EXAMPLE 1!

5 β-Cholestan-3-one was prepared using 4-cholesten-3-one (Aldrich Co.)as a starting material by the method of Tsuji et al. (N. Tsuji, J.Suzaki, and M. Shiota, J. Org. Chem., 45: 2729, 1980) (yield: 99%).

PREPARATION EXAMPLE 2!

Dienol acetate was synthesized by the method of Chowdhury et al. (P. K.Chowdhury, R. P. Sharma, and J. N. Barua, Tetrahedron Lett., 24: 3383,1983) as follows:

Trimethylsilyl chloride (5.5 ml) was added to a solution of4-cholesten-3-one (3.84 g, 10 mmol, Aldrich Co.) in an acetic anhydridesolution (40 ml). The mixture was refluxed under heating in argon gasfor 3 hours. After vacuum concentration, the residue was dissolved inethyl acetate, washed with a saturated aqueous solution of sodiumcarbonate and a saturated saline solution and dried over magnesiumsulfate. The solvents were distilled off and the residue was subjectedto silica gel column chromatography to yield dienol acetate (3.3 g,yield: 88%).

Then, cholesta-4,6-dien-3-one was prepared from the dienol acetate bythe method of Minami et al. (I. Minami, K. Takahashi, I. Shimizu, and J.Tsuji, Tetrahedron, 42: 2971, 1986) (yield: 83%).

PREPARATION EXAMPLE 3!

The dienol acetate prepared in Preparation Example 2 (3.77 g, 8.8 mmol)was dissolved in 140 ml of dichloromethane and a dioxane(70ml)-phosphate buffer (pH 7.0, 70 ml) was added. The mixture was thencooled at 0° C. and Br₂ (841 μl, 8.8 mmol) was added dropwise understirring. The resulting solution was stirred at 0° C. for 100 minutesand a mixture of an aqueous solution of sodium thiosulfate (1.5 g) andan aqueous solution of sodium bicarbonate (3.0 g) was then added. Theresulting mixture was stirred for additional 15 minutes. The organiclayer was separated, washed with a saturated aqueous solution of sodiumcarbonate and a saturated saline solution and dried over magnesiumsulfate. After the solvents were distilled off under vacuum, the residuewas subjected to silica gel column chromatography to yield 6β-bromo-4-cholesten-3-one (3.23 g, yield: 80%).

PREPARATION EXAMPLE 4!

Dichloromethane (50 ml) was added to pyridinium chlorochromate (2.78 g,12.9 mmol) and molecular shieves (3 A) (4.37 g) and the mixture wasstirred under a nitrogen atmosphere at room temperature for 15 minutes.To the resulting mixture was added a dichloromethane solution (30 ml) of5,6-epoxycholesterol (α:β=5:1) (3.323 g, 8.6 mmol) prepared by themethod of Fieser and Fieser (L. F. Fieser and H. Fieser, Reagents forOrganic Synthesis, Vol. 1: 136, 1967) and the mixture was stirred for 3hours. Ether (300 ml) was added to the reaction solution and theresulting solution was filtered through silica gel-magnesium sulfate.The organic layer was washed twice with 50 ml of a saturated salinesolution and dried. The solvents were distilled off and the residue waspurified by column chromatography. 6-Hydroxy-4-cholesten-3-one (α:β=5:1)was obtained as a colorless crystal from the hexane-ethyl acetate (5:1)eluate (yield: 51.7%).

PREPARATION EXAMPLE 5!

4-Cholesten-3,6-dione was prepared using 4-cholesten-3-one (Aldrich Co.)as a starting material by reference to the method of Hevl and Herr (F.W. Hevl and M. E. Herr, J. Am. Chem. Soc., 75: 1918, 1953) and that ofMalhotra et al. (S. K. Malhotra, J. J. Hostynek and A. F. Lundin, J. Am.Chem. Soc., 90: 6565, 1968). Stated specifically, pyrrolidine (2.85 g)was added to a benzene solution (50 ml) of 4-cholesten-3-one (3.84 g)and the mixture was refluxed under heating for 24 hours. After cooling,the solvent and the excess pyrrolidine were distilled off under vacuum.Upon recrystallization from methanol, a colorless crystal of dienamine(4.16 g) was obtained. Cupric acetate (100 mg) was added to abenzene-methanol (500:10) solution of the dienamine (4.16 g) and air wasintroduced into the mixture for 24 hours. Then, a 2% aqueous solution(20 ml) of acetic acid was added and the mixture was stirred for 30minutes and washed successively with water, a 1% sodium carbonatesolution and a saturated saline solution. The resulting residue wasdried, filtered and subjected to silica gel column chromatography(hexane:ethyl acetate=10:1) to yield a colorless crystal of4-cholestene-3,6-dione (3.23 g).

EXAMPLE 1! A. Test Method

5-Cholesten-3-one purchased from Sigma Co., 5 β-cholestan-3-one asprepared in Preparation Example 1, cholesta-4,6-dien-3-one as preparedin Preparation Example 2 and 6 β-bromo-4-cholesten-3-one as prepared inPreparation Example 3 were used as test compounds; and 4-cholesten-3-onepurchased from Aldrich Co. was used as a comparative compound. Thefollowing examination was carried out with these compounds.

1. Animals and Feeding Conditions

Six male CDF1 mice (5 weeks, BALB/c X DBA/2) purchased from JapaneseCharles River Co., Ltd. were used in each experimental group.

All the mice were divided into experimental groups and placed inaluminum cages (22 cm×33 cm×11 cm height). They were fed and allowed tofeed and water ad libitum at a temperature of 24±1° C. and a relativehumidity of 55±5% for 2 weeks in a laboratory where light and darknesswere controlled change every 12 hours. Each cage and floor mat (whiteflakes) were exchanged twice a week.

2. Preparation of Test Feeds

A synthetic feed (powder feed), or modified AIN (Oriental Yeast Co.),was used as a basic feed to prepare test feeds as described below in1)-6). The basic feed was composed of 22.8% proteins, 54.1%carbohydrates, 6.0% lipids, 4.9% fibers, 2.9% ash and 8.7% water(calorie: 1,523 KJ).

The addition of a test compound at 0.5 wt % to the basic feedcorresponds to the administration of the test compound at 995 mg/kg bodyweight/day per mouse on average. The test feeds were stored at 4° C.after preparation and a fresh one was daily provided.

1) Feed for 5-cholesten-3-one group: 5-Cholesten-3-one purchased fromSigma Co. was added to the basic feed at 0.5 wt %.

2) Feed for 5 β-cholestan-3-one group: 5 β-Cholestane-3-one was added tothe basic feed at 0.5 wt %.

3) Feed for cholesta-4,6-dien-3-one group: Cholesta-4,6-dien-3-one wasadded to the basic feed at 0.5 wt %.

4) Feed for 6 β-bromo-4-cholesten-3-one group: 6 β-Bromo-cholesten-3-onewas added to the basic feed at 0.5 wt %.

5) Feed for control-1 group: 4-Cholesten-3-one was added to the basicfeed at 0.5 wt %.

6) Feed for control-2 group: Nothing was added to the basic feed.

3. Test Items

The mice were weighed at given intervals of time and the average valuesof the various experimental groups were determined. In addition, thefeed consumptions were measured at given intervals of time and theaverage feed efficiencies (increase in body weight per 100 g of feed) ofthe various experimental groups were determined. After the end of thefeeding, all the mice were anesthetized to death with carbon dioxide gasand autopsied. The brains, lungs, hearts, livers, spleens, kidneys,testis, adrenals and intraperitoneal lipids were weighed and the averagevalues of the various experimental groups were determined.

B. Test Results

(1) Measurement of the change in body weight and the feed consumption

The changes in the average body weights of the various experimentalgroups are shown in FIG. 1. In FIG. 1, *, ** and *** show the presenceof a significant difference from the non-treated control-2 group atprobability (p) levels of <5%, <1% and <0.1%, respectively, in a t-test.

The increase in body weight was greatly inhibited in the5-cholesten-3-one group, 5 β-cholestan-3-one group,cholesta-4,6-dien-3-one group and 6 β-bromo-4-cholesten-3-one group,compared to the non-treated control-2 group. The inhibition of theincrease in body weight in these groups was also greater than in thecontrol-1 group treated with 4-cholesten-3-one. In particular, the5-cholesten-3-one group showed a remarkable inhibition of the increasein body weight. The average body weight of the 5-cholesten-3-one groupdecreased up to the 10th day of feeding, increased a little at day 14and leveled off thereafter.

In conclusion, the test compounds, 5-cholesten-3-one, 5β-cholestan-3-one, cholesta-4,6-dien-3-one and 6β-bromo-4-cholesten-3-one are more effective in inhibiting the increasein body weight than the comparative compound 4-cholesten-3-one.

The average feed efficiencies of the various experimental groups areshown in Table 1. Table 1 shows clearly that the groups with the lowerincrease in body weight had lower feed efficiencies. Therefore, it wasfound that the inhibitory effect of the test compounds on the increasein body weight was not due to the diminution of appetite.

                  TABLE 1                                                         ______________________________________                                        Feed Efficiencies of CDF1 Mice                                                Experimental Group    1 week  2 week                                          ______________________________________                                        1. 5-Cholestan-3-one group                                                                          -3.32   -0.39                                           Cholestan-3-one group 3.53    6.11                                            3. Cholesta-4,6-dien-3-one group                                                                    5.13    5.77                                            bromo-4-cholesten-3-one group                                                                       5.49    4.48                                            5. Control-1 (4-cholesten-3-one) group                                                              5.85    5.99                                            6. Control-2 (non-treated) group                                                                    7.87    6.98                                            ______________________________________                                    

(2) Measurement of intraperitoneal lipid

The average intraperitoneal lipids in the various experimental groupsare shown in FIG. 2. The intraperitoneal lipid in all the mice treatedwith the test compounds was smaller with a statistically significantdifference than in the non-treated control-2 group. In FIG. 2, ** and*** show the presence of a significant difference from the non-treatedcontrol-2 group at probability (p) levels of <1% and <0.1%,respectively, in a t-test. The differences in intraperitoneal lipidbetween the test compound-treated groups and the non-treated control-2group were in substantial proportion to the levels of the inhibition ofthe increase in body weight. Therefore, it was found that theadministration of the test compounds results in the accumulation ofsmaller amounts of body lipids, which is one of the primary reasons forthe inhibition of the increase in body weight.

(3) Measurement of organ weights

The average organ weights are shown in Table 2. The values in Table 2are represented by relative weights per 100 g of body weight. Thesymbols *, ** and *** show the presence of a significant difference fromthe non-treated control-2 group at probability (p) levels of <5%, <1%and <0.1%, respectively, in a t-test. When the body weight decreases, adecrease in the amount of body lipid is followed by a decrease in theamount of muscles. On the other hand, the amounts of main organs whichfulfill important functions in the body will not decrease as much, sothe relative weights of the main organs will increase.

A relative increase in the weight of spleen was observed in the5-cholesten-3-one group, 5 β-cholestan-3-one group and 6β-bromo-4-cholesten-3-one group. In the 5-cholesten-3-one group, arelative increase in the weights of brain and testis was observed. Inthe 5 β-cholestan-3-one group, a relative decrease in the weight of lungwas also observed. In the control-1 group treated with4-cholesten-3-one, a relative increase in the weights of spleen andtestis was observed. Abnormal values were not recognized in any groups,suggesting that the test compounds have little or no toxicity and sideeffects.

                                      TABLE 2                                     __________________________________________________________________________    Relative Weights of Main Organs in CDF1 Mice.sup.a                            Experimental                                                                        Brain                                                                             Heart                                                                             Lung                                                                              Liver                                                                             Spleen                                                                            Kidney                                                                            Adrenal                                                                           Testis                                      Group (g) (g) (g) (g) (g) (g) (mg)                                                                              (g)                                         __________________________________________________________________________    1     1.86***                                                                           0.53                                                                              0.98                                                                              4.88                                                                              0.33**                                                                            1.39                                                                              17.17                                                                             0.76*                                       2     1.54                                                                              0.54                                                                              0.83*                                                                             4.24                                                                              0.37***                                                                           1.61                                                                              14.88                                                                             0.64                                        3     1.53                                                                              0.59                                                                              0.91                                                                              4.14                                                                              0.29                                                                              1.58                                                                              14.42                                                                             0.66                                        4     1.57                                                                              0.55                                                                              1.00                                                                              5.00                                                                              0.30*                                                                             1.54                                                                              14.70                                                                             0.66                                        5     1.48                                                                              0.51                                                                              0.87                                                                              4.01                                                                              0.37***                                                                           1.56                                                                              15.67                                                                             0.69*                                       6     1.47                                                                              0.54                                                                              0.98                                                                              4.50                                                                              0.26                                                                              1.63                                                                              16.62                                                                             0.59                                        __________________________________________________________________________     .sup.a, the numerical values are the average for six mice in each group. 

None of the experimental groups had aberrations such as abnormalities inthe colors of the main organs and the occurrence of tumor. All the miceseemed to be normal to the naked eye.

In regard to general appearance, the 5-cholesten-3-one group mice had asign of emaciation but mice in the other groups showed no abnormalitiesin the color of hair and behavior. They also did not develop anyabnormal symptoms such as diarrhea.

In conclusion, the results of measurement of the main organ weights andthe autopsical finding with the naked eye demonstrated that the testcompounds have little of no side effects or toxicity.

EXAMPLE 2! A. Test Method

A test feed was prepared by adding 0.5 wt % of a test compound(6-hydroxy-4-cholesten-3-one as prepared in Preparation Example 4) to abasic feed, or modified AIN (Oriental Yeast Co.). Feeds for control-1and -2 groups were prepared by the same method as in Example 1. Theother procedures were the same as in Example 1.

B. Test Results

(1) Change in body weight

The changes in the average body weights of the various experimentalgroups are shown in FIG. 3. In FIG. 3, * shows the presence of asignificant difference from the non-treated control-2 group at aprobability (p) level of <5% in a t-test. The increase in body weightwas significantly inhibited in the 6-hydroxy-4-cholesten-3-one group.After 14 days of the treatment, the increase in body weight of the6-hydroxy-4-cholesten-3-one group was significantly different from thatof the non-treated control-2 group and smaller than that of the positivecontrol 4-cholesten-3-one group (control-1 group).

In conclusion, the test compound 6-hydroxy-4-cholesten-3-one is moreeffective in inhibiting the increase in body weight than the comparativecompound, 4-cholesten-3-one.

(2) Change in feed consumption

The feed efficiencies of the various experimental groups are shown inTable 3. The feed efficiencies of the various experimental groups weresubstantially consistent with the tendency of increasing body weight.Therefore, it was confirmed that the inhibitory effect of the6-hydroxy-4-cholesten-3-one on the increase in body weight was due tothe reduction in feed efficiency rather than to the decrease in feedintake.

                  TABLE 3                                                         ______________________________________                                        Feed Efficiencies of CDF1 Mice                                                Experimental Group    1 week  2 week                                          ______________________________________                                        1. 6-hydroxy-4-cholesten-3-one group                                                                1.76    3.29                                            2. Control-1 (4-cholesten-3-one) group                                                              4.68    5.84                                            3. Control-2 (non-treated) group                                                                    5.52    7.64                                            ______________________________________                                    

(3) Measurement of intraperitoneal lipid

The average intraperitoneal lipids in the various experimental groupsare shown in FIG. 4. In FIG. 4, * and *** show the presence of asignificant difference from the non-treated control-2 group atprobability (p) levels of <5% and <0.1%, respectively, in a t-test. Theintraperitoneal lipid in the 6-hydroxy-4-cholesten-3-one group wassignificantly smaller than in the non-treated control-2 group.Therefore, it was found that 6-hydroxy-4-cholesten-3-one is effective ininhibiting the accumulation of body lipids, particularly intraperitoneallipids.

(4) General conditions and autopsical finding

The general conditions of the 6-hydroxy-4-cholesten-3-one group were asnormal as in the non-treated control-2 group in terms of the color ofhair, feces, behavior and appetite. In anatomical examination after thetreatment, no pathological abnormalities in organs were observed withthe naked eye. The organ weights (relative weights per 100 g of bodyweight) of the various experimental groups are shown in Table 4. InTable 4, *, ** and *** show the presence of a significant differencefrom the non-treated control-2 group at probability (p) levels of <5%,<1% and <0.1%, respectively, in a t-test.

The weights of the brain, liver and spleen in the6-hydroxy-4-cholesten-3-one group were a little greater than in thenon-treated control-2 group. However, in the absence of any pathologicalchanges in these organs, it was verified that there was no toxicinfluence.

In conclusion, it was shown that 6-hydroxy-4-cholesten-3-one has littleor no toxicity.

                                      TABLE 4                                     __________________________________________________________________________    Relative Weights of Main Organs in CDF1 Mice.sup.a                            Experimental                                                                        Brain                                                                             Heart                                                                             Lung                                                                              Liver                                                                             Spleen                                                                            Kidney                                                                            Adrenal                                                                           Testis                                      Group (g) (g) (g) (g) (g) (g) (mg)                                                                              (g)                                         __________________________________________________________________________    1     1.72**                                                                            0.55                                                                              0.74                                                                              5.00**                                                                            0.33**                                                                            1.49                                                                              11.53                                                                             0.70                                        2     1.63                                                                              0.55                                                                              0.78                                                                              4.90                                                                              0.38***                                                                           1.65                                                                              11.83                                                                             0.71                                        3     1.58                                                                              0.53                                                                              0.65                                                                              4.73                                                                              0.27                                                                              1.66                                                                              15.39                                                                             0.60                                        __________________________________________________________________________     .sup.a, the numerical values are the average for six mice in each group. 

The results demonstrated that 6-hydroxy-4-cholesten-3-one exhibits ananti-obesity effect by inhibiting the accumulation of body lipids andthat it has little or no toxicity.

EXAMPLE 3! A. Test Method

A test feed was prepared by adding 0.5 wt % of a test compound(4-cholestene-3,6-dione as prepared in Preparation Example 5) to a basicfeed, or modified AIN (Oriental Yeast Co.). Feeds for control-1 and -2groups were prepared by the same method as in Example 1. The otherprocedures were the same as in Example 1.

B. Test Results

(1) Change in body weight

The changes in the average body weights of the various experimentalgroups are shown in FIG. 5. In FIG. 5, *** shows the presence of asignificant difference from the non-treated control-2 group at aprobability (p) level of <0.1% in a t-test. The increase in body weightwas significantly inhibited in the 4-cholestene-3,6-dione group. After 4days of the treatment, the body weight of the 4-cholestene-3,6-dionegroup was significantly different from that of the non-treated control-2group and smaller than that of the positive control 4-cholesten-3-onegroup (control-1 group).

In conclusion, the test compound 4-cholestene-3,6-dione is moreeffective in inhibiting the increase in body weight than the comparativecompound 4-cholesten-3-one.

(2) Change in feed consumption

The feed efficiencies of the various experimental groups are shown inTable 5. The feed efficiencies of the various experimental groups weresubstantially consistent with the tendency of increasing body weight.Therefore, it was confirmed that the inhibitory effect of4-cholestene-3,6-dione on the increase in body weight was due to thereduction in feed efficiency rather than to the decrease in feed intake.

                  TABLE 5                                                         ______________________________________                                        Feed Efficiencies of CDF1 Mice                                                Experimental Group    1 week  2 week                                          ______________________________________                                        1. 4-Choelstene-3,6-dione group                                                                     -7.24   -3.28                                           2. Control-1 (4-cholesten-3-one) group                                                              6.40    4.04                                            3. Control-2 (non-treated) group                                                                    8.77    10.17                                           ______________________________________                                    

(3) Measurement of intraperitoneal lipid

The average intraperitoneal lipids in the various experimental groupsare shown in FIG. 6. In FIG. 6, * and *** show the presence of asignificant difference from the non-treated control-2 group atprobability (p) levels of <5% and <0.1%, respectively, in a t-test. Theintraperitoneal lipid in the 4-cholestene-3,6-dione group wassignificantly smaller than that in the non-treated control-2 group andthe effect of 4-cholestene-3,6-dione was stronger than that of4-cholesten-3-one. Therefore, it was found that 4-cholestene-3,6-dioneis more effective in inhibiting the accumulation of body lipids,particularly intraperitoneal lipids.

(4) General conditions and autopsical finding

The general conditions of the 4-cholestene-3,6-dione group were asnormal as in the non-treated control-2 group in terms of the color ofhair, feces, behavior and appetite. In anatomical examination after thetreatment, no pathological abnormalities in organs were observed withthe naked eye. The organ weights (relative weights per 100 g of bodyweight) of the various experimental groups are shown in Table 6. InTable 6, ** and *** show the presence of a significant difference fromthe non-treated control-2 group at probability (p) levels of <1% and<0.1%, respectively, in a t-test.

The weights of the brain, kidney and testis in the4-cholestene-3,6-dione group had significant differences from thenon-treated control-2 group but the differences were very small. Inaddition, in the absence of any pathological changes in these organs, itwas verified that there was no toxic influence.

In conclusion, it was shown that 4-cholestene-3,6-dione has little or notoxicity.

                                      TABLE 6                                     __________________________________________________________________________    Relative Weights of Main Organs in CDF1 Mice.sup.a                            Experimental                                                                        Brain                                                                             Heart                                                                             Lung                                                                              Liver                                                                             Spleen                                                                            Kidney                                                                            Adrenal                                                                           Testis                                      Group (g) (g) (g) (g) (g) (g) (mg)                                                                              (g)                                         __________________________________________________________________________    1     2.05**                                                                            0.60                                                                              0.70                                                                              4.67                                                                              0.32                                                                              1.31**                                                                            13.35                                                                             0.87**                                      2     1.58                                                                              0.52                                                                              0.82                                                                              4.60**                                                                            0.31                                                                              1.59                                                                              16.37**                                                                           0.63                                        3     1.50                                                                              0.56                                                                              0.76                                                                              4.86                                                                              0.29                                                                              1.64                                                                              10.60                                                                             0.65                                        __________________________________________________________________________     .sup.a, the numerical values are the average for six mice in each group. 

EXAMPLE 4! A. Test Method

5-Cholesten-3-one purchased from Sigma Co. was used as a test compoundand 4-cholesten-3-one purchased from Aldrich Co. was used as acomparative compound. The following examination was carried out withthese compounds.

1. Animals and Feeding Conditions

Three male Sprague-Dawley (SD) rats (4 weeks) purchased from JapaneseCharles River Co., Ltd. were used in each experimental group.

2. Preparation of Test Feeds

A test feed was prepared by adding 0.5 wt % of a test compound(5-cholesten-3-one) to a basic feed, or modified AIN (Oriental YeastCo.). A feed for control-1 group was prepared by the same method asabove except that the comparative compound 4-cholesten-3-one was usedinstead of 5-cholesten-3-one.

3. The other procedures were the same as in Example 1.

B. Test Results

(1) Change in body weight and intraperitoneal lipid

At day 14 after the treatment, there was no significant difference inbody weight between the experimental groups. However, theintraperitoneal lipid in the 5-cholesten-3-one group was smaller thanthat in the 4-cholesten-3-one group (control-1 group) as shown in FIG.7.

(2) General conditions and autopsical finding

The general conditions of the 5-cholesten-3-one group were as normal asin the 4-cholesten-3-one group (control-1 group) in terms of the colorof hair, feces, behavior and appetite.

The organ weights (relative weights per 100 g of body weight) of theexperimental groups are shown in Table 7. In Table 7, * and ** show thepresence of a significant difference from the non-treated control-2group at probability (p) levels of <5% and <1%, respectively, in at-test. In the group of the comparative compound 4-cholesten-3-one, asignificant enlargement of the adrenal, as well as congestion andswelling of the spleen were observed. In contrast, the 5-cholesten-3-onegroup did not show any more abnormalities in the main organs than thenon-treated control-2 group. Although there were significant differencesin the lung weight of the 4-cholesten-3-one group and the brain weightsof the 4-cholesten-3-one group and the 5-cholesten-3-one group in at-test, the differences were small and within the normal range,therefore, they were not considered to have been caused by any toxiceffects.

                                      TABLE 7                                     __________________________________________________________________________    Relative Weights of Main Organs in SD Rats.sup.b                              Experimental                                                                        Brain                                                                             Heart                                                                             Lung                                                                              Liver                                                                             Spleen                                                                            Kidney                                                                            Adrenal                                                                           Testis                                      Group (g) (g) (g) (g) (g) (g) (mg)                                                                              (g)                                         __________________________________________________________________________    1     0.77**                                                                            0.37                                                                              0.65                                                                              5.10                                                                              0.27                                                                              0.94                                                                              13.63                                                                             0.85                                        2     0.74*                                                                             0.39                                                                              0.64*                                                                             5.05                                                                              0.33*                                                                             0.95                                                                              26.90**                                                                           0.95                                        3     0.81                                                                              0.41                                                                              0.60                                                                              5.02                                                                              0.26                                                                              0.99                                                                              13.39                                                                             0.92                                        __________________________________________________________________________     .sup.b, the numerical values are the average for three rats in each group     Experimental Group 1: 5Cholesten-3-one group                                  Experimental Group 2: Control1 (4cholesten-3-one) group                       Experimental Group 3: Control2 (nontreated) group                        

As described above, the comparative compound 4-cholesten-3-one exhibitsside effects on SD rats, such as an enlargement of the adrenal, as wellas congestion and swelling of the spleen; on the other hand,5-cholesten-3-one does not exhibit any of these side effects and hencehas weaker toxicity than 4-cholesten-3-one.

INDUSTRIAL APPLICABILITY

The anti-obesity agents of the present invention can inhibit theincrease in body weight and the accumulation of body lipids. They areuseful due to the little or no side effects and toxicity. Therefore, theanti-obesity agents of the present invention can be used to inhibit theaccumulation of body lipids, thereby preventing obesity.

The anti-obesity agents of the present invention can be used to treatobesity, thereby keeping appropriate levels of body weight.

The anti-obesity agents of the present invention can be used to preventand/or treat various diseases associated with obesity, such as diabetes,hypertension, arteriosclerosis, hyperlipemia, cardiac diseases,nephropathy, gout, fatty liver, gallstone, sleep apnea, osteoarthritis,emmeniopathy, sterility, mammary carcinoma, uterus carcinoma, cancer ofrectum, prostatic cancer and the like.

The anti-obesity agents of the present invention can also be used toprevent and/or treat mental disorders associated with obesity, such asbulimia, cibophobia and the like.

The anti-obesity agents of the present invention can also be used toprevent and/or treat obesity and disorders associated with obesity inpet animals such as dogs, cats and the like.

The anti-obesity agents of the present invention can even be used toinhibit the excess accumulation of body lipids in domestic animals andcultured fish, thereby improving the quality of their flesh.

We claim:
 1. An anti-obesity agent which comprises at least one compoundselected from the group consisting of 5-cholesten-3-one,5,β-cholestan-3-one, cholesta-4,6-dien-3-one,6,β-bromo-4-cholesten-3-one, 6-hydroxy-4-cholesten-3-one and4-cholestene-3,6-dione as an active ingredient.
 2. The anti-obesityagent of claim 1, wherein the 3-ketosteroid compound is5-cholesten-3-one.
 3. The anti-obesity agent of claim 1, wherein the3-ketosteroid compound is 5 β-cholestan-3-one.
 4. The anti-obesity agentof claim 1, wherein the 3-ketosteroid compound ischolesta-4,6-dien-3-one.
 5. The anti-obesity agent of claim 1, whereinthe 3-ketosteroid compound is 6 β-bromo-4-cholesten-3-one.
 6. Theanti-obesity agent of claim 1, wherein the 3-ketosteroid compound is6-hydroxy-4-cholesten-3-one.
 7. The anti-obesity agent of claim 1,wherein the 3-ketosteroid compound is 4-cholestene-3,6-dione.
 8. Apharmaceutical composition which comprises selected from the groupconsisting of 5-cholesten-3-one, 5,β-cholestan-3-one,cholesta-4,6-dien-3-one, 6,β-bromo-4-cholesten-3-one,6-hydroxy-4-cholesten-3-one and 4-cholestene-3,6-dione as an activeingredient and a pharmaceutically acceptable carrier.
 9. A method forpreventing obesity which comprises administering an effective amount ofat least one compound selected from the group consisting of5-cholesten-3-one, 5,β-cholestan-3-one, cholesta-4,6-dien-3-one,6,β-bromo-4-cholesten-3-one, 6-hydroxy-4-cholesten-3-one and4-cholestene-3,6-dione to a human.
 10. A method for treating obesitywhich comprises administering an effective amount of at least onecompound selected from the group consisting of 5-cholesten-3-one,5,β-cholestan-3-one, cholesta-4,6-dien-3-one,6,β-bromo-4,cholesten-3-one, 6-hydroxy-4-cholesten-3-one and4-cholestene-3,6-dione to a human.